Plasma Membrane Cholesterol Content Affects Nitric Oxide Diffusion Dynamics and Signaling

Abstract

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO ϳ20 -40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser 239 induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.As a small sized gaseous free radical, nitric oxide (NO) 2 presents unique challenges toward understanding the nature of its signaling in biological systems. Numerous levels of regulation at the level of nitric oxide synthase catalysis influence the rate of NO generation. Spatial localization of the biological signal is secondarily determined by a combination of the distinct physical and chemical properties of NO within the context of the microenvironment in which it was formed. The lifetime of NO molecules is governed chiefly by their relative abundance in relation to other radicals (1, 2), transition metal centers (3, 4), and oxygen (O 2 ) (5, 6). In addition to its very small size, the diffusional path of NO from the point of origin is affected by its preferentially hydrophobic solubility (7,8), resulting in an enrichment of NO in biological membranes relative to the aqueous milieu. In the present work, the hypothesis that cells may utilize plasma membrane cholesterol to further orchestrate spatial heterogeneity in NO biological signaling activity was tested.Cholesterol, a major lipid component of the plasma membrane in eukaryotic cells, plays an essential role in maintaining membrane fluidity and architecture (9 -11). Cells tightly control the ratio ...

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